Opening small packages: unraveling roles for microproteins during early vertebrate development

打开小包装：揭示微生物蛋白在早期脊椎动物发育过程中的作用

基本信息

批准号：
10678492
负责人：
Anthony J Treichel
金额：
$ 3.17万
依托单位：
STOWERS INSTITUTE FOR MEDICAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678492
关键词：
Affect Amino Acids Base Sequence Biochemical Bioinformatics Biology Catalogs Cell division Cell physiology Cells Classification Clustered Regularly Interspaced Short Palindromic Repeats Computational Biology Congenital Abnormality Coupled Data Data Set Development Developmental Biology Diagnostic Disease Down-Regulation Embryo Embryonic Development Epitopes Etiology Exclusion Exhibits Gene Expression Genes Genetic Genetic Transcription Genome Germ Layers Goals Guide RNA Hour Human Human Development Image Immunity Immunofluorescence Immunologic Investigation Link Masks Mass Spectrum Analysis Maternal Messenger RNA Messenger RNA Microscopy Modeling Molecular Molecular Biology Molecular Genetics Open Reading Frames Pathway interactions Pattern Phenotype Play Population Positioning Attribute Process Protein Biochemistry Proteins Proteomics Publishing Quality of life RNA RNA Interference RNA Sequences Role Signal Transduction Specific qualifier value System Techniques Testing Therapeutic Training Translating Translations Untranslated RNA Untranslated Regions Variant Zebrafish cardiogenesis cell growth cell motility comparative developmental disease experimental study gastrulation gene function grasp heart function human disease human embryonic stem cell image guided imaging approach improved in vivo innovation insight knock-down migration mutant novel offspring pluripotency protein function ribosome profiling skills spatiotemporal transcription factor transcriptome sequencing zebrafish development zygote

项目摘要

Project Summary Human development relies on highly coordinated cell division, signaling, migration, and differentiation. Any perturbations of the protein effectors that orchestrate these critical processes can lead to human birth defects and/or diseases1-3. While our classical catalog contains around 20,000 proteins, evidence from ‘omics-based techniques has generated a rapid paradigm shift in RNA biology4-12. Notably, RNA sequences defined as non- coding in fact produce short proteins (£ 100 amino acids) called microproteins that modulate diverse processes13- 40 including heart function26, immunity22,27,41, and cell growth29,30. For example, a microprotein called APELA maintains pluripotency in human embryonic stem cells20 and is critical for zebrafish heart development16,19. However, additional microprotein function(s) during vertebrate development remain largely unknown. Zebrafish is an outstanding model for interrogating vertebrate gene function. Their genetic tractability coupled with external, synchronous development is well-suited for developmental analyses. Further, hundreds of microproteins have been identified across zebrafish development using ribosome profiling, mass- spectrometry, and conservation analyses16,42. Remarkably, APELA is the only microprotein out of these 400 that is currently characterized. A key barrier to further microprotein investigation is that a majority of messenger RNAs (mRNA) during early development are maternally provided and can mask the effects of a targeted gene disruption. Our novel CRISPR/Cas13d system43,44 overcomes this barrier because it actively degrades its target mRNA and therefore enables selective knockdown of maternally provided mRNAs in zebrafish. My preliminary experiments with CRISPR/Cas13d have revealed that knockdown of one microprotein mRNA inhibits zygotic genome activation and disrupts posterior patterning. This study will combine CRISPR/Cas13d and ‘omics-based techniques to interrogate microprotein function during zebrafish development. Aim 1 will leverage CRISPR/Cas13d to elucidate microproteins important for early development. Then, Aim 2 will determine the cell and molecular processes that rely on developmental microproteins. Together, these aims will define and characterize a population of microproteins involved in vertebrate development. Experimental approaches will develop my skills in bioinformatics, molecular genetics, developmental biology, and protein biochemistry. Microproteins critical for zebrafish development will be informative for expanding the catalog of human proteins through comparative analyses. Further, microproteins with functions during development represent uncharted therapeutic and/or diagnostic opportunities for human birth defects and/or human diseases with developmental origins.

项目摘要人类发展依赖于高度协调的细胞分裂，信号传导，迁移和分化。编排这些关键过程的蛋白质效应的任何扰动都会导致人类的先天缺陷和/或疾病1-3。虽然我们的古典目录包含约20,000种蛋白质，但“基于OMICS的证据技术在RNA生物学4-12中产生了快速的范式转移。值得注意的是，RNA序列定义为非 - 实际上，编码产生了称为微蛋白的短蛋白（100英镑的氨基酸），可调节各种过程13- 40包括心脏功能26，免疫学22,27,41和细胞生长29,30。例如，一种称为apela的微蛋白维持人类胚胎干细胞中的多能性20，对于斑马鱼心发育至关重要16,19。但是，脊椎动物发育过程中的其他微蛋白功能在很大程度上未知。斑马鱼是询问脊椎动物基因功能的出色模型。他们的遗传障碍结合外部，同步开发非常适合发展分析。此外，数百个使用核糖体分析，质量 - 光谱法和保护分析16,42。值得注意的是，APELA是这400种中唯一的微蛋白目前是特征。进一步的微蛋白投资的关键障碍是大多数Messenger RNA （mRNA）在早期开发过程中主要提供，并且可以掩盖靶向基因的影响破坏。我们的小说CRISPR/CAS13D System43,44克服了这一障碍，因为它会积极降低其目标 mRNA，因此可以选择性地敲除斑马鱼中的母体提供的mRNA。我的初步 CRISPR/CAS13D的实验表明，一个微蛋白mRNA的敲低抑制了zygotic 基因组激活并破坏后模式。这项研究将结合CRISPR/CAS13D和“基于OMICS的技术来询问微蛋白斑马鱼发育过程中的功能。 AIM 1将利用CRISPR/CAS13D来阐明微蛋白很重要为了早期发展。然后，AIM 2将确定依赖发育的细胞和分子过程微蛋白。这些目标共同定义并表征了涉及的微蛋白群脊椎动物的发展。实验方法将发展我在生物信息学，分子遗传学方面的技能，发育生物学和蛋白质生物化学。对于斑马鱼开发至关重要的微蛋白将是通过比较分析来扩展人类蛋白质目录的信息。此外，微蛋白在开发过程中的功能代表了人类的未知疗法和/或诊断机会具有发育起源的先天缺陷和/或人类疾病。